發(fā)布時間：2018-01-27 08:26

  本文關(guān)鍵詞： 磺胺氯吡嗪鈉 單克隆抗體 化學(xué)發(fā)光免疫檢測 磺胺噻唑　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：磺胺氯吡嗪鈉(sulfachlorpyrazine sodium,SPZ)是磺胺類藥物的一種,在獸醫(yī)臨床上用來治療家禽球蟲病。若大劑量使用或不遵守休藥期規(guī)定,藥物易在動物體內(nèi)蓄積,威脅人類健康。免疫學(xué)檢測方法作為獸藥殘留大規(guī)模檢測的主要方法,具有特異性強(qiáng),靈敏度高等特點(diǎn)。目前國內(nèi)外關(guān)于SPZ殘留的酶聯(lián)免疫檢測方法(ELISA)的報(bào)道較少,未見關(guān)于SPZ的化學(xué)發(fā)光免疫分析(CLIA)檢測方法的報(bào)道。本文制備了SPZ的單克隆抗體,在此基礎(chǔ)上建立CLIA方法;同時對磺胺藥物多殘留快速檢測技術(shù)進(jìn)行探索,合成了藥物的母核抗原-磺胺噻唑(TS)并制備其多克隆抗體,分別用ELISA方法和CLIA方法對抗體進(jìn)行鑒定。1、SPZ單克隆抗體的制備及鑒定。本實(shí)驗(yàn)通過重氮化方法制備了完全抗原SPZ-OVA,免疫小鼠4次后,選取血清抗體效價較好的小鼠脾細(xì)胞進(jìn)行細(xì)胞融合。經(jīng)過篩選并將陽性克隆按有限稀釋法亞克隆3次,得到穩(wěn)定分泌抗體的細(xì)胞株。將細(xì)胞株擴(kuò)大培養(yǎng)后,腹腔注射小鼠制備腹水。將腹水單克隆抗體D9純化后通過間接ELISA確定包被原最佳濃度為0.01μg/m L,單抗的工作濃度為1:4×105,在最佳抗原抗體工作濃度下進(jìn)行ci-ELISA實(shí)驗(yàn),建立標(biāo)準(zhǔn)曲線,得到曲線線性方程為y=-0.3369x+1.347,R2=0.994,計(jì)算得抗體半數(shù)抑制濃度(IC50)為326ng/m L;與其他測試藥物的交叉反應(yīng)小于5%,表明單抗特異性良好。2、SPZ化學(xué)發(fā)光免疫檢測(CLIA)方法的建立。在單克隆抗體Mab D9的基礎(chǔ)上建立SPZ的CLIA方法,在0.02-4μg/m L的范圍內(nèi),SPZ濃度的對數(shù)值與相對發(fā)光值呈線性關(guān)系,線性方程為y=-0.3602x+1.2874,R2=0.981,IC50值為151ng/m L;標(biāo)準(zhǔn)曲線的批內(nèi)變異系數(shù)為7.02%,批間變異系數(shù)為8.5%;雞肉樣品最低檢測限為36.3ng/g,樣品添加回收率在75%-85%之間。3、磺胺類藥物族特異性抗體的制備。首先合成磺胺母核結(jié)構(gòu)TS,然后用碳二亞胺(EDC)法合成免疫原TS-BSA,將其免疫小鼠后,對抗血清進(jìn)行篩選,得到兩只小鼠血清效價分別為1:2.56×105和1:1.28×105;選用效價高的小鼠血清進(jìn)一步檢測。經(jīng)ci-ELISA實(shí)驗(yàn)和CLIA實(shí)驗(yàn)測得抗體對TS的IC50值分別為33ng/m L和7.9ng/m L;與供試磺胺藥物中含有五元雜環(huán)的藥物有較強(qiáng)交叉反應(yīng)(CR26%);與部分六元雜環(huán)藥物有一定的交叉反應(yīng)(1%CR11%);與磺胺氯吡嗪鈉(SPZ)和磺胺二甲嘧啶鈉(SM2)無交叉反應(yīng)(CR0.01%)。實(shí)驗(yàn)表明抗體與多種R基結(jié)構(gòu)的SAs可產(chǎn)生交叉反應(yīng)。
[Abstract]:Sulfadiazine sodium sulfadiazine is a kind of sulfonamides. Used in veterinary clinic for the treatment of coccidiosis in poultry. If the drug is used in large doses or does not comply with the withdrawal period, the drug is easily accumulated in the animal body. As the main method of large-scale detection of veterinary drug residues, immunological detection has strong specificity. The characteristics of high sensitivity. At present, there are few reports at home and abroad about the Elisa method of SPZ residue detection. There was no report on the chemiluminescence immunoassay of SPZ. The monoclonal antibody against SPZ was prepared and the CLIA method was established. At the same time, the rapid detection technique of sulfadiazine multiresidue was explored, and the mother nucleus antigen-sulfathiazolium (TS) of the drug was synthesized and its polyclonal antibody was prepared. ELISA method and CLIA method were used to identify the monoclonal antibody. The complete antigen SPZ-OVA was prepared by diazotization. After the mice were immunized for 4 times, the spleen cells with good antibody titers were selected for cell fusion, and the positive clones were subcloned by limited dilution method for 3 times. The cell lines secreting antibodies stably were obtained. Ascites were prepared by intraperitoneal injection of mice. After purification of monoclonal antibody D9 in ascites, indirect ELISA was used to determine the optimal concentration of the coating material was 0. 01 渭 g / mL, and the working concentration of the monoclonal antibody was 1: 4 脳 10 ~ 5. The ci-ELISA test was carried out at the best working concentration of antigens and antibodies, and the standard curve was established. The linear equation of the curve was yang-0.3369x 1.347R2O0.994. IC50 was calculated to be 326ng / mL; The cross-reaction with other tested drugs was less than 5%, indicating that the specificity of the McAb was good (.2%). The establishment of SPZ chemiluminescence immunoassay (CLIA). Based on the monoclonal antibody Mab D9, the CLIA method of SPZ was established in the range of 0.02-4 渭 g / mL. The logarithmic value of SPZ concentration is linearly related to the relative luminescence value. The linear equation is YCU -0.3602x 1.2874N R2O 0.981N IC50 = 151ng / mL; The coefficient of variation in the standard curve was 7.02 and the coefficient of variation between batches was 8.5. The minimum detection limit of chicken samples was 36.3 ng / g. The recovery of the samples was between 75% and 85%. The preparation of sulfonamides group specific antibodies was performed. First, the mother nuclear structure TS of sulfanilamide was synthesized. Then the immunogen TS-BSAwas synthesized by carbodiimide EDCmethod and then immunized with TS-BSA. the antiserum was screened. The titers of two mice were 1: 2.56 脳 105 and 1: 1.28 脳 105, respectively. Ci-ELISA test and CLIA test showed that the IC50 value of antibody to TS was 33ng / mL and 7.9 ng / mL, respectively. There was a strong cross reaction between CR26 and sulfamethylamine containing five heterocyclic compounds. There was a certain cross reaction with some hexagonal heterocyclic drugs. There was no cross reaction with sulfamethazine (SSP) and sulfadimethazine sodium (SM2). The results showed that the antibody could cross react with SAs with various R-group structures.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.79

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本文編號：1468062